基于酵母雙雜交的EgSmadE表達載體構建及自激活檢測
發(fā)布時間：2018-10-22 07:39
【摘要】：目的構建細粒棘球蚴SmadE(Echinococcos granulosus SmadE)基因的酵母雙雜交真核表達載體,并檢測其表達重組蛋白的毒性和自激活活性。方法以PCR技術獲得EgSmadE基因片段,并克隆至酵母雙雜交載體pGADT7及pGBKT7。應用PEG/LiAc法將重組載體導入Y2HGold酵母感受態(tài)細胞,利用固體培養(yǎng)基表型篩選對其毒性和自激活活性進行檢測。結果擴增EgSmadE基因全長1 129bp,與酵母雙雜交重組載體連接后獲得pGADT7-EgSmadE和pGBKT7-EgSmadE,經PCR、限制性內切酶雙酶切和測序均正確;連接產物轉化酵母菌后經培養(yǎng)基表型鑒定與對照組菌落大小一致,表明重組載體表達產物對酵母菌無毒性;SD/-Trp和SD/-Leu培養(yǎng)基均有白色菌落生長,而SD/-Leu/X/A和SD/-Trp/X/A無菌落生長,表明重組載體表達產物對下游報告基因無自激活活性。結論成功構建酵母雙雜交載體pGADT7-EgSmadE及pGBKT7-EgSmadE,可用于鑒定EgSmadE蛋白質,為宿主與棘球蚴相互作用的分子機制研究奠定基礎。
[Abstract]:Objective to construct the yeast two-hybrid eukaryotic expression vector of SmadE (Echinococcos granulosus SmadE) gene of Echinococcus granulosus, and to detect the toxicity and self-activation activity of the recombinant protein. Methods EgSmadE gene fragment was obtained by PCR and cloned into yeast two-hybrid vector pGADT7 and pGBKT7.. The recombinant vector was introduced into Y2HGold yeast receptive cells by PEG/LiAc method, and its toxicity and self-activation activity were detected by phenotypic screening of solid medium. Results the total length of EgSmadE gene was 1129bp. after being ligated with yeast two-hybrid recombinant vector, pGADT7-EgSmadE and pGBKT7-EgSmadE, were digested and sequenced correctly by PCR, restriction endonuclease. The results showed that the recombinant vector expression product was not toxic to yeast, and the white colony growth was found in SD/-Trp and SD/-Leu medium, while SD/-Leu/X/A and SD/-Trp/X/A had no colony growth, indicating that the recombinant vector expression product had no self-activating activity to the downstream reporter gene. Conclusion the successful construction of yeast two-hybrid vector pGADT7-EgSmadE and pGBKT7-EgSmadE, can be used to identify EgSmadE protein and lay a foundation for the study of molecular mechanism of interaction between host and echinococcus.
【作者單位】： 新疆醫(yī)科大學基礎醫(yī)學院生理學教研室;新疆醫(yī)科大學第一附屬醫(yī)院/臨床醫(yī)學研究院 新疆包蟲病基礎醫(yī)學重點實驗室;
【基金】：新疆維吾爾自治區(qū)自然科學基金項目(No.2015211C029)
【分類號】：R532.32
[Abstract]:Objective to construct the yeast two-hybrid eukaryotic expression vector of SmadE (Echinococcos granulosus SmadE) gene of Echinococcus granulosus, and to detect the toxicity and self-activation activity of the recombinant protein. Methods EgSmadE gene fragment was obtained by PCR and cloned into yeast two-hybrid vector pGADT7 and pGBKT7.. The recombinant vector was introduced into Y2HGold yeast receptive cells by PEG/LiAc method, and its toxicity and self-activation activity were detected by phenotypic screening of solid medium. Results the total length of EgSmadE gene was 1129bp. after being ligated with yeast two-hybrid recombinant vector, pGADT7-EgSmadE and pGBKT7-EgSmadE, were digested and sequenced correctly by PCR, restriction endonuclease. The results showed that the recombinant vector expression product was not toxic to yeast, and the white colony growth was found in SD/-Trp and SD/-Leu medium, while SD/-Leu/X/A and SD/-Trp/X/A had no colony growth, indicating that the recombinant vector expression product had no self-activating activity to the downstream reporter gene. Conclusion the successful construction of yeast two-hybrid vector pGADT7-EgSmadE and pGBKT7-EgSmadE, can be used to identify EgSmadE protein and lay a foundation for the study of molecular mechanism of interaction between host and echinococcus.
【作者單位】： 新疆醫(yī)科大學基礎醫(yī)學院生理學教研室;新疆醫(yī)科大學第一附屬醫(yī)院/臨床醫(yī)學研究院 新疆包蟲病基礎醫(yī)學重點實驗室;
【基金】：新疆維吾爾自治區(qū)自然科學基金項目(No.2015211C029)
【分類號】：R532.32
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